Immunoglobulins that are plasma proteins containing antibodies against various viruses and bacteria are used as drugs to prevent or treat diseases by administration to either subjects who naturally lack antibodies or patients who are in need of artificial supplement of antibodies because of viral or bacterial diseases.
In order to use such immunoglobulins as drugs, immunoglobulins for subcutaneous or intramuscular injection have been prepared according to the cold ethanol fractionation process (Cohn E. et al., J. Am. Chem. Soc., 68:459, 1946) developed by Cohn and Oncley or the modified cold ethanol fractionation process (Kistler P, Nitschmann H S, Vox Sang, 7:414. 1952) developed by Kistler and Nitschmann.
However, immunoglobulins for intramuscular injection have the following problems: 1) the doses of such immunoglobulins are limited, making it impossible to administer the immunoglobulins in large amounts; 2) the immunoglobulins cause pain at the site injected with the immunoglobulins; 3) the immunoglobulins have a low content of natural immunoglobulin G (IgG) having antibody activity; 4) the antibody activity of the immunoglobulins is reduced by protease at the injected site; and 5) the time taken to reach peak blood concentrations is 24 hours or more.
In order to solve the problems of intramuscular injection, administration of immunoglobulins by intravenous injection was attempted. However, when immunoglobulin preparations were administered intravenously, a variety of immediate side effects, including difficult breathing and circulatory system shock, appeared due to a serious side effect (anaphylactic reaction) attributable to aggregates with anti-complementary activity. Such symptoms appeared mainly in immunoglobulin-deficient patients. Particularly, a side effect of serious hypersensitivity was observed in patients in which anti-IgA antibodies appeared.
In other words, in order to develop an intravenous formulation, impurities such as blood clotting factors (fibrinogen, albumin, prekallikrein activator (PKA) and transferrin) or immunoglobulins similar to hepatitis B immunoglobulin such as IgA and IgE should be removed, and it is necessary to increase the stability and purity.
Thus, as intravenous injection of immunoglobulins is impossible due to the above-described problems, development of immunoglobulin preparations for intravenous injection has been required, and methods capable of removing the above-described aggregates and/or preventing aggregate formation during preparation processes have been developed. Intravenous injection of immunoglobulins has become possible as a result of treating immunoglobulins with proteases such as pepsin, papain or Plasmin, or chemical substances such as β-propiolactone, to change their structure so as to suppress the formation of immunoglobulin aggregates or destroy immunoglobulin aggregates, thereby reducing the anti-complementary activities of the immunoglobulins.
The first-generation intravenous immunoglobulin (IVIG) products were prepared by treating a starting material (Cohn fraction II) with pepsin to remove immunoglobulin aggregates. The preparation process did not comprise a column chromatography step, and the prepared product was lyophilized so as to be stably maintained over a suitable period of time, and was dissolved immediately before use. However, it was found that IVIG products manufactured by some manufacturers caused viral infections such as viral hepatitis C. For this reason, one or more steps of inactivating and/or removing known virus were added to the preparation process. Thereafter, the second-generation IVIG products with low anti-complementary activity and higher stability were disclosed in the mid-1980s, and the IVIG products were purified by several chromatography steps.
Such preparations were injected intravenously, and thus overcame the disadvantages of intramuscular immunoglobulins, including limited dose, pain at the injected site, and the reduction in antibody of immunoglobulins by protease, and the time taken to reach peak blood concentrations was also reduced to several hours or less.
However, the intravenous immunoglobulin products as described above have little or no natural IgG with antibody activity due to their structural change, and thus have reduced or no complement binding ability and also have a blood half-life as short as about 4-12 days, suggesting that they exhibit no satisfactory effects on the prevention and treatment of diseases. Furthermore, the first-generation and second-generation IVIG products prepared in the form of lyophilized powder require an additional process for dissolving them, and have low dissolution rates. For this reason, liquid IVIG products have been developed, and improved processes have been required to obtain more stable and pure IVIG products.
In connection with this, German Patent No. 2,604,759 and U.S. Pat. No. 4,124,576 discloses methods of obtaining pure IgG (third-generation IVIG) with antibody activity by using a non-ionic surfactant such as polyethylene glycol, unlike the above-described gamma-immunoglobulin for intravenous injection. Such IgG preparations have complement binding ability and increased blood half-lives, and thus can show good effects on the prevention and treatment of diseases. However, these preparations produced by treatment with polyethylene glycol can still cause side effects, because it is difficult to completely remove aggregates with anti-complementary activity from these preparations (showing an anti-complementary activity of about 0.02 U/mg).
In addition, Korean Patent No. 1983-0007083 discloses a method of preparing an intravenous immunoglobulin from Cohn fraction II or fraction II+III, isolated from human plasma, by treatment with polyethylene glycol. However, there are problems in that the process is complicated and the yield is low.
Meanwhile, hepatitis B virus (HBV) is a virus with a DNA genome, which belongs to the Hepadnaviridae family and causes acute and chronic hepatitis. Hepatitis B virus (HBV) is classified into eight genotypes having a difference of about 8% or more in the gene nucleotide sequence, or it is classified into four serotypes (adw, adr, ayw and ayr, etc.) based on the two antigenic determinants (d/y and w/r) of hepatitis B surface antigen (HBsAg). About 3.5 hundred million people worldwide have chronic hepatitis B virus (HBV) infection. Particularly, in Korea and China, people with chronic hepatitis B virus infection reach about 5-8%, and hepatitis B virus (HBV) infection is the major cause of liver disease and liver cancer. Currently developed vaccines can be somewhat effective in the prevention of hepatitis B virus infection, but a significant number of patients with chronic hepatitis B virus infection still exist. Chronic infection with hepatitis B virus (HBV) causes hepatitis, cirrhosis and liver cancer, and the incidence of liver cancer is about 300 times higher in people with chronic hepatitis B virus infection than in non-infected people. According to the WHO report, about 80% of liver cancer is caused by chronic hepatitis B.
Currently known therapeutic agents for hepatitis B include the nucleoside analogues including lamivudine and adefovir dipivoxil, which inhibit the DNA replication of hepatitis B virus (HBV) by inhibiting the reverse transcriptase of hepatitis B virus polymerase (HBV polymerase). However, when these drugs are administered for 3 years, drug-resistant virus occurs in about 75% of the patients, thereby reducing the therapeutic effect of the drug. Due to this problem, it is impossible to treat hepatitis B infection using the viral replication inhibitors alone. For this reason, it was attempted to use these inhibitors in combination with interferon agents, but these inhibitors are not currently used due to serious side effects.
For a similar therapeutic purpose, a hepatitis B immune globulin (HBIG) preparation comprising a hepatitis B virus (HBV) antibody isolated and purified from blood having a high antibody titer was taken into consideration. However, because the antibody of the HBIG preparation is isolated and purified from plasma, there are problems, including difficulty in obtaining plasma, the possibility of viral infection, low activity, high costs and the like.
The present inventors have made extensive efforts to solve the above-described problems occurring in the prior art, and as a result, have found that thrombogenic substances in plasma fraction are effectively removed and the stability of an immunoglobulin product is improved, by dialyzing and concentrating fraction II isolated from plasma, and then purifying the fraction by anion exchange chromatography and cation exchange chromatography while controlling the salt concentration during cation exchange chromatography and elution, thereby completing the present invention.